This invention relates to biologically active vitamin D.sub.2 compounds. More specifically, this invention relates to the hormonally active, natural metabolite 1.alpha.,24(S)-dihydroxy vitamin D.sub.2 and to methods of preparing this metabolite and the nonbiological epimer 1.alpha.,24(R)-dihydroxy vitamin D.sub.2. This invention also relates to a pharmaceutical composition which includes a pharmaceutically effective amount of 1.alpha.,24(S)-dihydroxy vitamin D.sub.2, and to a method of controlling abnormal calcium metabolism by administering a pharmaceutically effective amount of the compound.
Vitamin D and its active metabolites are known to be important in regulating calcium metabolism in animals and humans. The naturally occurring form of vitamin D in animals and humans is vitamin D.sub.3. It has been shown that in animals, including humans, vitamin D.sub.3 is activated by being hydroxylated in the C.sub.25 position in the liver, followed by 1.alpha.-hydroxylation in the kidney to produce the hormone 1.alpha.,25-dihydroxy vitamin D.sub.3 ["1.alpha.,25-(OH).sub.2 D.sub.3 "]. See, U.S. Pat. No. 3,880,894. The major physiological pathway for catabolism of the vitamin D.sub.3 metabolites, 25-hydroxy vitamin D.sub.3 and 1.alpha.,25-(OH).sub.2 D.sub.3, is initiated by C.sub.24 -oxidation. Holick, M. F., Kleiner-Bossallier, A., Schnoes, H. K., Kasten, P. M., Boyle, I. T., and DeLuca, H. F., J. Biol. Chem., 248, 6691-6696 (1973).
Vitamin D.sub.2 is the major, naturally occurring form of vitamin D found in plants. Vitamin D.sub.2 differs structurally from vitamin D.sub.3 in that vitamin D.sub.2 has a methyl group at C.sub.24 and has a double bond between C.sub.22 and C.sub.23.
Shortly after their discovery, it seemed apparent that vitamin D.sub.3 and vitamin D.sub.2 had similar, if not equivalent, biological activity. It has also been commonly believed that the metabolism (i.e., the activation and catabolism) of vitamin D.sub.2 was the same as for vitamin D.sub.3. See, Harrison's Principles of Internal Medicine: Part Seven, "Disorders of Bone and Mineral Metabolism: Chap. 35," in E. Braunwald, K. J. Isselbacher, R. G. Petersdorf, J. D. Wilson, J. B. Martin and H. S. Fauci (eds.), Calcium, Phosphorus and Bone Metabolism: Calcium Regulating Hormones, McGraw-Hill, New York, pp. 1860-1865. In this regard, the active form of vitamin D.sub.2 is believed to be 1.alpha.,25-dihydroxy vitamin D.sub.2 ["1.alpha.,25-(OH).sub.2 D.sub.2 "]. Further, 24-hydroxy derivatives of 25-hydroxy vitamin D.sub.2 and 1.alpha.,25-(OH).sub.2 D.sub.2, that is, 24,25-dihydroxy vitamin D.sub.2 and 1.alpha.,24,25-trihydroxy vitamin D.sub.2, are known, suggesting that catabolism of vitamin D.sub.2, like vitamin D.sub.3, proceeds through the same C.sub.24 oxidation step. Jones, G., Rosenthal, D., Segev, D., Mazur, Y., Frolow, F., Halfon, Y., Robinavich, D. and Shakked, Z., Biochemistry, 18:1094-1101 (1979).
It has recently been found, however, that an active analogue of vitamin D.sub.2, 1.alpha.-hydroxy vitamin D.sub.2 ["1.alpha.-(OH)D.sub.2 "] has pharmacological properties distinctly different than those exhibited by its vitamin D.sub.3 counterpart, 1.alpha.-hydroxy vitamin D.sub.3 ["1.alpha.-(OH)D.sub.3 "]. U.S. Pat. No. 5,104,864 discloses that 1.alpha.-(OH)D.sub.2 will reverse the loss of bone mass in human osteoporotic patients when administered at dosages of 2.0 .mu.g/day or higher. Because of toxicity, dosage levels of 2.0 .mu.g/day or greater are not safely obtained with 1.alpha.-(OH)D.sub.3.
Such distinct pharmacological properties may be explained fully, or in part, by the present inventors' discovery that pharmacological dosages of 1.alpha.-(OH)D.sub.2 administered to humans are metabolized in part to biologically active 1.alpha.,24(S)-dihydroxy vitamin D.sub.2 ["1.alpha.,24(S)--(OH).sub.2 D.sub.2 "]. As explained in more detail below, the hydroxylation at the carbon-24 position of the 1 -hydroxylated vitamin D.sub.2 molecule, represents an activation pathway peculiar to the vitamin D.sub.2 molecule.
While 1.alpha.,24(S)-dihydroxy vitamin D.sub.3 and 1.alpha.,24(R)-dihydroxy vitamin D.sub.3 ["1.alpha.,24(R/S)--(OH).sub.2 D.sub.3 "] have been chemically synthesized (U.S. Pat. No. 4,022,891) it has not been demonstrated that either is a natural compound found in biological systems. Furthermore, the present inventors have discovered that 1.alpha.,24(S)--(OH).sub.2 D.sub.2 has distinctly different biological activity from that exhibited by 1.alpha.,24(R/S)--(OH).sub.2 D.sub.3. For example, Ishizuka et al. have found that 1.alpha.,24(R)--(OH).sub.2 D.sub.3 binds the 1,25-(OH).sub.2 D.sub.3 receptor site more tightly than does 1,25-(OH).sub.2 D.sub.3 itself. Ishizuka, S., Bannai, K., Naruchi, T. and Hashimoto, Y., Steroids, 37:1,33-42 (1981); Ishizuka, S., Bannai, K., Naruchi, T. and Hashimoto, Y., Steroids, 39:1,53-62 (1982). Using a similar assay, the present inventors have discovered that the 1.alpha.,24(S)--(OH).sub.2 D.sub.2 is two-fold less competitive in binding the 1,25-(OH).sub.2 D.sub.3 receptor site than is 1,25-(OH).sub.2 D.sub.3. The present inventors have also found that 1.alpha.,24(S)--(OH).sub.2 D.sub.2 shows a relatively poor binding affinity for the vitamin D serum binding protein which is evidence of a rather short half life indicative of low toxicity.
The present inventors have demonstrated the presence of circulating 1.alpha.,24(S)--(OH).sub.2 D.sub.2 in humans administered 1.alpha.-(OH)D.sub.2. This indicates that in animals and man, vitamin D.sub.2 is naturally metabolized to both 1.alpha.,25-(OH).sub.2 D.sub.2 and 1.alpha.,24(S)--(OH).sub.2 D.sub.2. The relative ratios of the two vitamin D.sub.2 hormones appear to vary according to the precursor and the amount of precursor presented to the C.sub.24 pathway. Thus it appears that as dosages of 1.alpha.-(OH)D.sub.2 are increased, the ratio of 1.alpha.,24(S)--(OH).sub.2 D.sub.2 to 1.alpha.,25-(OH).sub.2 D.sub.2 increases.
These results which are presented in more detail below, indicate that 1.alpha.,24(S)--(OH).sub.2 D.sub.2 has the desirable characteristic of high biological activity ith low toxicity. The fact that 1.alpha.,24(S)--(OH).sub.2 D.sub.2 is a significant metabolite when pharmacological levels of 1.alpha.-(OH)D.sub.2 are administered indicates that 1.alpha.,24(S)--(OH).sub.2 D.sub.2 may be mediating the desirable pharmacological effects of 1.alpha.-(OH)D.sub.2 and is a useful therapeutic drug for treating various types of disorders involving calcium metabolism.